Nucleosomes and other components of chromatin can repress transcription by blocking the access of transcription factors and other regulatory proteins to DNA. Interactions between nucleosomes and linker histones promote the formation of 30 nm fibers and increasingly compact forms of higher-order chromatin structure, further limiting the access of regulatory proteins to DNA. Alterations in chromatin structure lead to a variety of human diseases, including cancer and birth defects. Although tremendous progress has been made toward determining the mechanism of action of chromatin-remodeling factors and histone-modifying enzymes, much remains to be learned about how they regulate transcription by altering the structure and spacing of nucleosomes. Even less is known about how higher-order chromatin structure is regulated and used to control gene expression in eukaryotic cells. To address these important issues, our laboratory uses Drosophila melanogaster as a model organism to study the function of chromatin-remodeling and modifying enzymes. Much of our work has been focused on the roles of Polycomb and trithorax group in transcription and development. Polycomb group proteins play important roles in the regulation of cellular pluripotency and differentiation by methylating lysine 27 of histone H3 over broad chromatin domains. This chromatin modification plays a key role in epigenetic gene silencing in organisms ranging from flies to humans. A key issue concerns how Polycomb group repression is overcome to permit the expression of genes required for differentiation. Recent studies in our lab have suggested that Kismet (KIS), an ATP-dependent chromatin-remodeling factor, plays an important role in this process by preventing the spread of H3K27 methylation into active genes. To test this hypothesis, we will compare the genome-wide distributions of KIS and H3K27 methylation and determine if KIS acts as a barrier to the spread of the repressive modification. Other studies in our laboratory have suggested that another chromatin-remodeling factor, Imitation-SWI (ISWI), plays a global role in chromatin compaction and transcriptional repression by promoting the association of the linker histone H1 with chromatin. To test this hypothesis, we will compare changes in gene expression resulting from the loss of ISWI and histone H1 function in vivo. We will also use complementary genetic, biochemical and cell biological approaches to determine how ISWI promotes the association of histone H1 with chromatin and identify other factors involved in this process. By studying multiple chromatin- remodeling factors in a single model organism, we will gain a much better understanding of their roles in transcription, development and disease.